A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells.
In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the beta-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9 x 10(-4), whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8 x 10(-4). The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.
Bärtsch
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Department of Genetics, Swiss Federal Institute of Technology (ETH) Zürich, Switzerland.
Würgler
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FE
Sengstag
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C
D000818:Animals; D002478:Cells, Cultured; D002875:Chromosomes; D004331:Drosophila melanogaster / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005434:Flow Cytometry; D017930:Genes, Reporter; D005839:Gentamicins / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000633:toxicity; D016685:Mitomycin / Q000633:toxicity; D008938:Mitosis; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic / Q000187:drug effects*; D012091:Repetitive Sequences, Nucleic Acid; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D005839:Gentamicins; D009153:Mutagens; D016685:Mitomycin; D005020:Ethyl Methanesulfonate; C010680:antibiotic G 418; D008741:Methyl Methanesulfonate; D001616:beta-Galactosidase